Genomic DNA was isolated from patient EDTA blood using the DNeasy Blood and Tissue Kit (Qiagen). TCR beta chain (TCRB) deep sequencing was performed to detect rearranged TCRβ gene sequences using hsTCRB Kit (Adaptive Biotechnologies) according to the manufacturer’s protocol. The prepared library was sequenced on an Illumina MiSeq by the Genomics & Proteomics Core Facility, German Cancer Research Center (DKFZ). Data processing (demultiplexing, trimming, gene mapping) was done using the Adaptive Biotechnologies proprietary platform. Data were visualized using the Treemap Visualization package version 2.4.2 (https://cran.r-project.org/web/packages/treemap/index.html ). TCRB sequencing data are available at https://clients.adaptivebiotech.com/pub/platten-2021-nature .
Hstcrb kit
Manufactured by Adaptive Biotechnologies
The HsTCRB Kit is a laboratory equipment product designed for the detection and analysis of T-cell receptor beta chain (TCRB) sequences. It provides a standardized workflow for the amplification and sequencing of TCRB gene segments, allowing researchers to study T-cell receptor diversity and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Immuneaccess platform, by Adaptive Biotechnologies (1 mentions)
Immunoseq analyzer 3, by Adaptive Biotechnologies (1 mentions)
Allprep dna rna kit, by Qiagen (1 mentions)
Immunoseq platform, by Adaptive Biotechnologies (1 mentions)
4 protocols using hstcrb kit
1
TCR Profiling of Patient Blood
2
TCR Repertoire Analysis of TIL Cultures
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For analysis of the TCR repertoire, DNA was isolated from tumor tissue and expanded TIL cultures using the NucleoSpin Tissue Kit (Macherey-Nagel). TCRB deep sequencing was performed to detect rearranged TCRB gene sequences using the hsTCRB Kit (Adaptive Biotechnologies) according to the manufacturer’s protocol. Prepared libraries were sequenced on an Illumina MiSeq by the Genomics & Proteomics Core Facility, German Cancer Research Center (DKFZ). Data processing (demultiplexing, trimming, and gene mapping) was done using Adaptive Biotechnologies´ proprietary platform. The data was converted and analyzed using VDJtools version 1.2.1. Treemaps for visualization of the data were generated using the treemap R-package (R version 4.0.3).
3
Clonal T-cell Receptor Sequencing
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For sequencing of T-cell receptors (TCRs) to determine the clonality of TCPs, DNA was isolated using an All-Prep DNA/RNA Kit (Qiagen) following the manufacturer's instructions. TCRβ sequencing was performed using a hsTCRb Kit (Adaptive Biotechnologies) following the manufacturer's instructions and analyzed with the corresponding ImmunoSEQ-Analyzer 3.0 software. Briefly, the most variable complementary-determining region 3 (CDR3), spanning the recombination site of V-D-J recombinations of TCR β-chains was sequenced. Productive rearrangements were regarded as unique in-frame nucleotide sequences without stop codon, leading to a functional TCR. Productive frequency means the individual frequency of a specific productive rearrangement (clone) among all productive rearrangements. Clonality was calculated based on productive entropy normalized to the total number of productive rearrangements. Sample overlap was investigated using the Morisita index considering unique clones, individual frequencies of clones and the probability of a common origin of two samples. TCR sequencing data is accessible at the ImmuneACCESS platform http://adaptivebiotech.com/pub/amini-2019-frontimmunol (Adaptive Biotechnologies).
4
Bulk TCRβ Sequencing for Vaccine Studies
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Bulk TCRβ sequencing was undertaken on 12 study participants for both the 2016-17 and 2017-18 vaccination seasons for the d0 and d7 populations using the Adaptive Biotechnologies hsTCRb kit (Adaptive Biotechnologies, Seattle, Washington). Briefly, genomic DNA was extracted from the bulk sorted cells using DNA IQ (Promega). A multiplex PCR-based method was used to target and amplify multiple TCR targets with amplification bias accounted for with internal templates (61 (link)). Resultant templates were sequenced on the Illumina MiSeq, performed by the Vanderbilt VANTAGE DNA sequencing core. Sequences were analysed using the ImmunoSEQ platform (Adaptive Biotechnologies) and RStudio (62 ). TCRβ sequencing was performed at Survey level resolution (i.e. at a lower sequencing depth that is optimal for samples with low numbers of T cells and more clonal samples). Only TCR sequences unique to each subject at the nucleotide level were included in the final analyses. This conservative approach was utilized as it was not possible to distinguish between public clonotypes or contamination by rare TCR sequences. Numbers of unique and total TCRβ sequences for each subject and population are presented in Table S5
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